Cytotype diversity and genome size variation in eastern Asian polyploid Cardamine (Brassicaceae) species

Abstract

Background and aims: Intraspecific ploidy-level variation is an important aspect of a species' genetic make-up, which may lend insight into its evolutionary history and future potential. The present study explores this phenomenon in a group of eastern Asian Cardamine species.

Methods: Plant material was sampled from 59 localities in Japan and Korea, which were used in karyological (chromosome counting) and flow cytometric analyses. The absolute nuclear DNA content (in pg) was measured using propidium iodide and the relative nuclear DNA content (in arbitrary units) was measured using 4,6-diamidino-2-phenylindole fluorochrome.

Key results: Substantial cytotype diversity was found, with strikingly different distribution patterns between the species. Two cytotypes were found in C. torrentis sensu lato (4x and 8x, in C. valida and C. torrentis sensu stricto, respectively), which displays a north-south geographical pattern in Japan. Hypotheses regarding their origin and colonization history in the Japanese archipelago are discussed. In Korean C. amaraeiformis, only tetraploids were found, and these populations may in fact belong to C. valida. C. yezoensis was found to harbour as many as six cytotypes in Japan, ranging from hexa- to dodecaploids. Ploidy levels do not show any obvious geographical pattern; populations with mixed ploidy levels, containing two to four cytotypes, are frequently observed throughout the range. C. schinziana, an endemic of Hokkaido, has hexa- and octoploid populations. Previous chromosome records are also revised, showing that they are largely based on misidentified material or misinterpreted names.

Conclusions: Sampling of multiple populations and utilization of the efficient flow cytometric approach allowed the detection of large-scale variation in ploidy levels and genome size variation attributable to aneuploidy. These data will be essential in further phylogenetic and evolutionary studies.

Fig. 1.

(A) Distribution of cytotypes of Cardamine torrentis s.l. (comprising C. torrentis s.s. and C. valida), C. schinziana and C. amaraeiformis in Japan and South Korea. (B) Distribution of cytotypes of C. yezoensis in Hokkaido and northern Honshu. Intra-population cytotype variation is illustrated by two- to four-coloured symbols. Type localities (locus classicus) of relevant names are indicated by species epithets.

Fig. 2.

Mitotic metaphase chromosomes. (A) Cardamine yezoensis, 2n = 48 (sample JP 46). (B) C. yezoensis, 2n = 72 (sample JP 120). (C) C. schinziana, 2n = 48 (sample 07-811). (D) C. schinziana, 2n = 64 (sample JP 77). (E) C. torrentis s.l., 2n = 64 (= C. torrentis s.s., sample JP 96). (F) C. torrentis s.l., 2n = 64 (= C. torrentis s.s., sample JP 8904). (G) C. torrentis s.l., 2n = 32 (= C. valida, sample JP 116). (H) C. amaraeiformis, 2n = 33 (sample K 3). Scale bar = 5 µm. For details on localities, see Appendix.

Fig. 3.

(A–D) Flow-cytometric histograms of DAPI-stained nuclei obtained from different cytotypes of the studied Cardamine species and internal standards. Standards: (A,C) Lycopersicum esculentum as a primary internal standard; (B) Bellis perennis as a secondary internal standard; (D) sample JP 78-1 of C. schinziana as a secondary internal standard. Samples: (A) Cardamine torrentis s.l., 2n = 32 (= C. valida, population JP 117); (B) C. yezoensis, 2n = 80 (population JP 56); (C) C. amaraeiformis, 2n = 32 (population K 3); (D) C. schinziana, 2n = 64 (population JP 77). (E,F) Selected flow-cytometric histograms documenting intraspecific divergence in nuclear DNA content within ploidy levels. Two individuals of the same ploidy level but with different fluorescence intensities were analysed simultaneously (with DAPI-stained nuclei). (E) Cardamine torrentis s.l., 2n ≈ 4x (= C. valida, ind. JP 117-3 and JP 117-1: 6·58 % difference); (F) C. yezoensis, 2n ≈ 6x (ind. JP 54-4 and JP 47-3: 8·03 % difference). For details on localities see Appendix.